Mitochondrial movement is a feature of all eukaryotic cells. In neurons, the movements of mitochondria are particularly crucial because of the need to match the distribution of mitochondria to the energy requirements of subcellular regions. Axonal transport of mitochondria is thus required to ensure ATP production and Ca++ buffering at sites that are distant from the cell body. Moreover, the distribution of mitochondria will change in response to neural activity and the increased energy demands that activity places on the cell. The present proposal builds on our earlier studies in which we determined that a complex containing the proteins kinesin heavy chain, milton, miro, and O-GlcNAc transferase is needed for (+)-end directed movement of mitochondria. This proposal focuses on the regulation of mitochondrial movement and how it is mediated through members of this complex. Live imaging of mitochondria in rat hippocampal neurons and Drosophila axons is combined with biochemical and genetic studies so as to address mechanistic questions in vivo and in vitro. 1) We examine the significance of Ca++ regulation of mitochondrial motility via the EF hands of miro. 2) We examine the significance of the GTPase domains of miro to mitochondrial movement. 3) Another likely source of regulation is the post-translational modification of milton by O-GlcNAc transferase; this cytoplasmic glycosylating enzyme binds to milton and glycosylates it. We examine the significance of this modification for mitochondrial movement. 4) Finally, we ask how miro can also regulate retrograde movements of mitochondria. PUBLIC HEALTH RELEVANCE: Defects in mitochondrial distribution give rise to peripheral neuropathies including Charcot-Marie-Tooth Disease and may play a role in many neurodegenerative diseases. Our studies are designed to determine the normal means by which mitochondria are distributed in cells and thereby gain insights into potential pathological mechanisms, including the role of mitochondrial movement in responses to stress.